JOURNAL OF CLINICAL MICROBIOLOGY, June 2002, p. 2089–2094
0095-1137/02/$04.00?0 DOI: 10.1128/JCM.40.6.2089–2094.2002
Copyright © 2002, American Society for Microbiology. All Rights Reserved.
Vol. 40, No. 6
Detection of Replication-Competent Human Immunodeficiency Virus
Type 1 (HIV-1) in Cultures from Patients with Levels of HIV-1 RNA
in Plasma Suppressed to Less Than 500 or 50 Copies Per Milliliter†
Lisa M. Demeter,1* Ronald J. Bosch,2Robert W. Coombs,3Susan Fiscus,4James Bremer,5
Victoria A. Johnson,6Alejo Erice,7J. Brooks Jackson,8Stephen A. Spector,9Kathleen M. Squires,6‡
Margaret A. Fischl,10Michael D. Hughes,2and Scott M. Hammer11
University of Rochester School of Medicine and Dentistry, Rochester,1and Columbia University, New York,11New York; Harvard
School of Public Health, Boston, Massachusetts2; University of Washington, Seattle, Washington3; University of North Carolina,
Chapel Hill, North Carolina4; Rush Medical College, Chicago, Illinois5; University of Alabama at Birmingham School of
Medicine and Birmingham Veterans Affairs Medical Center, Birmingham, Alabama6; University of Minnesota,
Minneapolis, Minnesota7; Johns Hopkins University, Baltimore, Maryland8; University of California at
San Diego, San Diego, California9; and University of Miami School of Medicine, Miami, Florida10
Received 17 August 2001/Returned for modification 29 November 2001/Accepted 12 March 2002
We determined the frequency with which human immunodeficiency virus (HIV) peripheral blood mononu-
clear cell cultures convert from positive to negative in subjects enrolled in a substudy of AIDS Clinical Trials
Group (ACTG) 320, which compared the efficacy of treatment with a combination of indinavir, zidovudine, and
lamivudine (indinavir arm) to that of a combination of zidovudine and lamivudine (dual-nucleoside arm). All
subjects included for study had positive baseline HIV cultures. Cultures were performed in real time with 107
fresh patient peripheral blood mononuclear cells, using the ACTG consensus method. We found lower rates of
positive HIV cultures in the indinavir treatment arm than in the dual-nucleoside treatment arm (64 versus 96%
at week 24, P < 0.001). Within the indinavir arm of the study, we found that positive cultures were less likely
to occur in samples with a plasma HIV type 1 (HIV-1) RNA level of <500 copies/ml than in those with a level
of >500 copies/ml (44 versus 90%, P < 0.001). In addition, HIV cultures from samples with HIV-1 RNA levels
of >500 copies/ml turned positive 8.5 days earlier, on average, than those from samples with levels of <500
copies/ml (P < 0.001). However, 38% of samples with plasma RNA levels of <50 copies/ml still were positive
for HIV by culture. Thus, the rates of HIV isolation by standard culture procedures decrease as the plasma
viral load decreases below 1,000 copies/ml; however, HIV isolates were still obtained from a substantial
proportion of subjects with RNA levels of <50 copies/ml. The delay in the time required for HIV cultures to
turn positive should be considered when attempting to obtain an HIV isolate from patients with suppression
of plasma viral load.
Currently available combination antiretroviral regimens can
suppress human immunodeficiency virus type 1 (HIV-1) viral
load in plasma to below the limits of detection in a significant
proportion of patients (8, 9, 15). Initial reports of the inability,
with standard culture techniques, to culture HIV-1 from pa-
tients with viral load suppression raised the question of
whether circulating peripheral reservoirs of replication-com-
petent HIV-1 could be eradicated (13, 14, 16, 17). Subsequent
studies demonstrated that HIV-1 could be cultured from HIV-
infected patients with viral load suppression by using more
sensitive, labor-intensive techniques that included CD8 cell
depletion (2, 5, 13, 17). We performed real-time peripheral
blood mononuclear cell (PBMC) cultures using the AIDS Clin-
ical Trials Group (ACTG) consensus method in a subset of
subjects enrolled in a phase III clinical trial of indinavir,
zidovudine, and lamivudine. We found that this method, which
does not utilize CD8 depletion, resulted in the isolation of
HIV-1 in 38% of samples with plasma HIV-1 RNA levels that
were ?50 copies/ml. We have studied the impact of viral load
on the yield of HIV-1 cultures and have explored the ability of
a positive HIV culture to predict the subsequent viral load
MATERIALS AND METHODS
Study designs of ACTG 320 and ACTG 867. ACTG 320 was a phase III trial
of subjects with ?3 months of prior experience with zidovudine and CD4 cell
counts of ?200 cells/mm3, and it demonstrated that treatment with the combi-
nation of indinavir, zidovudine, and lamivudine (indinavir arm) was superior to
treatment with zidovudine and lamivudine (dual-nucleoside arm) in delaying
clinical progression (9). A total of 1,178 subjects had been randomized by 21
February 1997, the date that the study was terminated and the subjects were
unblinded, following an interim review that demonstrated an improved clinical
outcome in the indinavir arm of the study.
The primary goal of ACTG 867, the virology substudy of ACTG 320, was to
evaluate the correlations between HIV-1 RNA in plasma, qualitative PBMC
coculture, and syncytium-inducing (SI) phenotype. ACTG 867 was designed to
enroll a minimum of 200 subjects. Enrollment was limited to 8 of the 40 AIDS
Clinical Trials Units that participated in ACTG 320. Entry criteria for ACTG 867
were the same as those for ACTG 320 (9), except that subjects were asked to give
consent for the collection of additional blood samples for HIV-1 culture. In-
formed consent was obtained from all subjects, approval of study procedures was
obtained from all appropriate institutional review boards, and studies were
* Corresponding author. Mailing address: University of Rochester
Infectious Diseases Unit, 601 Elmwood Ave., Box 689, Rochester,
NY 14642. Phone: (585) 275-4764. Fax: (585) 442-9328. E-mail: Lisa
† AIDS Clinical Trials Group Protocol 867.
‡ Present address: LAC?USC Medical Center, Rand Schrader
Clinic, Los Angeles, CA 90033.
conducted in compliance with the appropriate federal guidelines. ACTG 867
enrolled 225 subjects between January and June 1996. Twenty-four subjects were
excluded from further analysis because no baseline culture result was available,
nine were excluded because they had a negative baseline culture, and two were
excluded because the baseline culture was indeterminate. An additional 15 pa-
tients were excluded because no follow-up HIV culture result was available at
any of the visits at weeks 8, 24, or 40. The remaining 175 subjects who had a
positive baseline culture and at least one follow-up PBMC culture (either posi-
tive or negative) were selected for further study. Follow-up was restricted to 40
weeks due to the early closure of the study, and the length of follow-up varied
among subjects according to the date of enrollment.
Virology studies. The plasma HIV-1 RNA concentration was determined by
using the AMPLICOR MONITOR assay (Roche Diagnostics, Branchburg,
N.J.). At the time these studies were conducted, preliminary data had demon-
strated a lower limit of quantification of 500 copies/ml for the standard AMPLI-
COR MONITOR assay. For this reason, all standard AMPLICOR results lower
than this limit were considered to be ?500 copies/ml. All samples with HIV-1
RNA concentrations of ?2,000 copies/ml according to the standard AMPLI-
COR MONITOR assay were retested using the Roche ultrasensitive assay, which
has a lower limit of quantification of 50 copies/ml. Six ACTG laboratories
performed batch assays after the conclusion of the clinical trial.
HIV-1 PBMC cultures were performed in real time in accordance with the
ACTG consensus assay (10). PBMCs from patients and HIV-negative donors
were isolated within 30 h of collection by Ficoll-Hypaque gradient centrifugation.
Before cocultivation with patient PBMCs, PBMCs from HIV-negative donors
were stimulated by culturing in RPMI 1640 with 20% heat-inactivated fetal
bovine serum, 100 U of penicillin per ml, 50 ?g of gentamicin per ml, 5%
interleukin-2, and 5 ?g of phytohemaglutinin (Difco) per ml at a concentration
of 2 ? 106cells/ml for 1 to 3 days at 37°C in 5% CO2. Patient and stimulated
donor PBMCs (107cells each) were then cocultivated in RPMI 1640 supple-
mented with 20% heat-inactivated fetal bovine serum, 2 mM L-glutamine, 100 U
of penicillin per ml, 50 ?g of gentamicin per ml, and 5% interleukin-2 (final
volume, 10 ml) at 37°C in 5% CO2. Patient and donor PBMCs used in this assay
were not CD8 cell depleted. Cultures were sampled for p24 antigen every 3 to 4
days and supplemented each week with fresh phytohemagglutinin- and interleu-
kin-2-stimulated PBMCs from HIV-negative donors. Negative cultures were
defined as those held for at least 18 days that had a supernatant p24 antigen
concentration of ?30 pg/ml. Cultures were defined as positive if at least one of
the following three criteria was met: two consecutive p24 values of greater than
30 pg/ml, of which the second value was at least four times greater than the first
value or had an optical density reading of ?2.0; two consecutive HIV p24 assay
values with an optical density reading of ?2.0; or three consecutive increasing
HIV p24 antigen values of ?30 pg/ml, where neither consecutive value was
greater than four times that of the previous samples but where the third value
was at least four times greater than the first. Cultures that had detectable p24
antigen without evidence for a sustained rise in concentration over time were
classified as indeterminate.
For analyses comparing plasma HIV-1 RNA concentrations with PBMC cul-
ture results, only results from the same date were used. Assays for SI phenotype
were performed at preentry and at week 40 by measuring the cytopathic effect of
each clinical HIV isolate in MT2 cells, according to previously published proce-
Statistical methods. The baseline HIV-1 RNA concentration was defined as
the geometric mean of the two measurements of HIV-1 RNA concentration
obtained prior to starting the study treatment. If either of these measurements
was outside the assay’s range of quantification, then an imputed value was used:
if a value of ?500 copies/ml was obtained (which occurred in 1% of subjects),
then a value of 500 copies was used, and if a value of ?750,000 copies/ml was
obtained (which occurred in 9% of subjects), then a value of 750,000 copies was
used. The baseline CD4 cell count was defined as the mean of the preentry and
entry measurements and excluded the screening value, which had to be ?200
cells/mm3. Analyses were based on subjects with available data, regardless of
whether they discontinued their randomized treatment. Fisher’s exact test and
the Cochran-Mantel-Haenszel (CMH) test (stratified by a screening CD4 cell
count of ?50 versus 51 to 200 cells/mm3) were used for comparison of propor-
tions. The Wilcoxon rank sum test was used for the comparison of ordered
categorical and continuously measured baseline variables. For tests involving the
number of days of culture, the generalized estimating equations (GEE) approach
was used to account for the possible correlation between multiple observations
for the same subject (12). Robust variance estimates with an exchangeable
working correlation were used. When treatment was tested, a covariate for the
stratification factor (screening CD4 cell count, ?50 versus 51 to 200 cells/mm3)
was included in the model. All analyses presented are based on randomized
treatment assignment. However, similar results were obtained when subjects
were analyzed in an as-treated fashion. Logistic regression analysis was used to
examine predictors of virologic suppression. Significance was assessed with the
likelihood ratio. All reported P values represent two-sided tests and are unad-
justed for multiple comparisons.
Patient population. The baseline characteristics of the study
subjects are shown in Table 1. The mean baseline CD4 cell
count was 83 cells/mm3; the baseline plasma HIV-1 RNA con-
centration was 4.95 log10copies/ml. The baseline characteris-
tics of subjects assigned to each of the two treatment arms were
similar, with the exception of a lower mean Karnofsky score for
subjects in the indinavir arm (89.4 versus 91.9, P ? 0.026,
Wilcoxon rank sum test). Overall, the baseline characteristics
of study subjects were similar to those of the subjects enrolled
in the parent study, ACTG 320 (9).
Plasma HIV-1 RNA responses according to treatment arm.
Suppression of plasma HIV-1 RNA levels to ?500 copies/ml
was achieved in 41% (34 of 83), 53% (41 of 77), and 56% (38
of 68) of subjects in the indinavir arm at weeks 8, 24, and 40,
respectively. In contrast, only 4% (4 of 89), 6% (5 of 81), and
15% (10 of 68) of subjects in the dual-nucleoside arm achieved
suppression of plasma HIV-1 RNA levels to ?500 copies/ml at
weeks 8, 24, and 40, respectively (P ? 0.001, CMH test at each
time point for comparisons between the two treatment arms).
TABLE 1. Baseline characteristics of the patient population
Value for patients
(n ? 90)
(n ? 85)
(n ? 175)
Sex, no. (%)
Age (yr), mean (SD)39.4 (8.8) 38.6 (7.5)39.0 (8.2)
Race and ethnicity, no. (%)
White and non-Hispanic
Black and non-Hispanic
Intravenous drug use,
Karnofsky score, mean (SD)91.9 (8.3) 89.4 (8.2)90.7 (8.3)
CD4 cell count (per mm3),
77.4 (57.1) 89.3 (66.6)83.2 (62.0)
No. of mo of prior ZDV
20 (8–39)22 (9–42) 21 (8–41)
HIV-1 RNA (log10copies/ml),
4.95 (0.68)4.94 (0.63) 4.95 (0.66)
aGroups were treated with zidovudine and lamivudine (ZDV?3TC) or with
indinavir, zidovudine, and lamivudine (IDV?ZDV?3TC).
2090 DEMETER ET AL.J. CLIN. MICROBIOL.
Suppression of plasma HIV-1 RNA levels to ?50 copies/ml
was achieved in 22% (18 of 83), 36% (27 of 76), and 46% (31
of 68) of subjects in the indinavir arm at weeks 8, 24, and 40,
respectively. Fewer than 7% of subjects in the dual-nucleoside
arm achieved suppression of plasma HIV-1 RNA levels to ?50
copies/ml at any time point (P ? 0.001, CMH test).
Frequency of positive PBMC cultures according to treat-
ment arm. There were significantly lower rates of positive
PBMC cultures after the initiation of therapy among subjects
in the indinavir arm than among subjects in the dual-nucleo-
side arm (P ? 0.001 at weeks 8, 24, and 40, CMH test) (Table
2). Although the duration of culture incubation beyond 18 days
was determined at the site level, this should not have led to
differences in the length of incubation of cultures between the
two arms of the study, since the sites and laboratories were
blinded as to treatment assignment and viral load responses.
We confirmed that duration of incubation for negative cultures
did not differ between the two treatment arms (P ? 0.78, GEE
test, including the CD4 cell count as a covariate).
Association of HIV-1 RNA concentration and PBMC culture
result. Because so few cultures from subjects in the dual-
nucleoside arm were negative, analysis of the association be-
tween plasma HIV-1 RNA concentration and PBMC culture
results was limited to samples obtained from subjects in the
indinavir arm. At each week, the proportion of subjects with
HIV-1 RNA concentrations of ?500 copies/ml who had posi-
tive PBMC cultures was lower than the proportion of subjects
with HIV-1 RNA concentrations of ?500 copies/ml (P ?
0.001, Fisher’s exact test) (Table 3).
Lower plasma HIV-1 RNA concentrations were associated
with a decreased likelihood of a positive PBMC culture. For
example, 95% of samples (81 of 85) with a plasma HIV-1 RNA
concentration of ?1,000 copies/ml by the standard AMPLI-
COR assay had a positive PBMC culture, compared with 65%
of those with 500 to 999 copies/ml and 44% of those with ?500
copies/ml (Table 4). This trend was also seen with the subset of
samples that were tested by the ultrasensitive assay (data not
Correlation of plasma HIV-1 RNA concentration with the
time required for PBMC cultures to become positive. We eval-
uated whether the apparently lower rate of culture isolation
from samples with low viral loads was due to these cultures
being incubated for a shorter period of time, leading to false-
negative results. Table 5 shows the duration of incubation of
the negative cultures obtained from subjects in the indinavir
arm, broken down according to plasma HIV-1 RNA concen-
tration. There was no significant difference in the incubation
times by plasma HIV-1 RNA concentration (P ? 0.32, GEE
However, there were differences in the times required for
TABLE 2. Frequency of positive HIV PBMC cultures,
by randomized treatment arma
No. of positive cultures/total no.
of cultures (%)a
ZDV?3TC armIDV?ZDV?3TC arm
aSubjects were treated with a combination of zidovudine and lamivudine
(ZDV?3TC arm) or indinavir, zidovudine, and lamivudine (IDV?ZDV?3TC
arm). Analyzed samples were obtained from each subject who had a positive
baseline PBMC culture and at least one follow-up culture (either positive or
bDetermined by use of the CMH test for the comparison between arms,
stratified by the screening CD4 cell count.
cExcludes one indeterminate culture.
TABLE 3. Frequency of positive HIV-1 PBMC cultures in subjects
randomized to receive triple therapy (including indinavir), according
to week of therapy and plasma HIV-1 RNA concentration
Frequency of positive PBMC culture in subjects
with indicated resultsa
Total 48/109 (44) 92/102 (90)28/73 (38)34/55 (62)
aNumber of subjects with positive HIV-1 PBMC cultures relative to the total
number of subjects. Values in parentheses are percentages.
bResults from seven cultures were excluded, due to missing plasma HIV-1
RNA results. The difference in the proportions of subjects who had positive
PBMC cultures was significant when comparing those with HIV-1 RNA levels of
?500 copies/ml and those with HIV-1 RNA levels of ?500 copies/ml at each
week (P ? 0.001, Fisher’s exact test).
cThe ultrasensitive assay was performed only on samples with a standard
AMPLICOR result of ?2,000 copies/ml.
dExcludes one culture which had indeterminate results.
TABLE 4. Frequency of positive cultures in subjects randomized to
receive triple therapy (indinavir arm) according to plasma HIV-1
RNA concentration (standard AMPLICOR assay)a
HIV-1 RNA concn
No. of positive cultures/
total no. of cultures
from samples (%)
?500.............................................................................. 48/109 (44)
500–999......................................................................... 11/17 (65)
1,000–4,999................................................................... 12/13 (92)
5,000–50,000................................................................. 37/40 (93)
?50,000......................................................................... 32/32 (100)
aContains only results obtained after baseline (week 8, 24, or 40).
TABLE 5. Distribution of negative cultures among ranges of
total incubation time, according to plasma HIV-1
RNA concentration in subjectsa
No. of days of
No. of negative cultures (cumulative %) at
HIV-1 RNA level ofb:
aSubjects were randomized to receive combination therapy with indinavir.
Results from three cultures are excluded because no plasma HIV-1 RNA result
was available for the same time at which the culture was performed.
bThere was no significant difference in the incubation times between the two
groups (P ? 0.32, GEE test).
VOL. 40, 2002HIV ISOLATION IN PATIENTS WITH VIRAL LOAD SUPPRESSION 2091
cultures to turn positive, depending on the plasma HIV-1 RNA
concentration of the sample used (Table 6). For example, 92%
of cultures from samples with plasma HIV-1 RNA concentra-
tions of ?500 copies/ml turned positive within the first 2 weeks
of incubation, compared to only 44% from samples with ?500
copies/ml. HIV-1 cultures from samples with plasma HIV-1
RNA concentrations of ?500 copies/ml turned positive 8.5
days earlier, on average, than cultures from samples with RNA
concentrations of ?500 copies/ml (P ? 0.001, GEE test) (Ta-
ble 6). Of the 103 cultures still negative at day 18, 32 (31%)
subsequently turned positive. All but 3 of these 32 cultures
were positive by day 28.
Predictors of virologic response. Our previous analyses of
the ACTG 320 cohort had shown that greater age, lower RNA
concentrations (baseline and week 8), and higher CD4 cell
counts (baseline and week 8) were independent predictors of
the virologic outcome at week 24 (3). Neither race and ethnic-
ity nor duration of prior zidovudine use were predictors of the
virologic outcome in those analyses. Similar associations were
seen in the subset of subjects described here (data not shown).
Because culture data for these subjects were also available,
we examined whether having a baseline SI virus isolate or a
positive PBMC culture at week 8 predicted the virologic out-
come at week 24. A baseline SI phenotype was not a significant
predictor of suppression of plasma HIV-1 RNA levels to ?500
copies/ml at week 24 or 40, although the small sample size may
have limited our ability to detect a significant association. For
example, in the 46 subjects who had SI virus at baseline, 57%
had HIV-1 RNA levels of ?500 copies/ml at week 24, com-
pared with 54% of the 28 subjects who had non-SI virus at
Among the 74 subjects in the indinavir arm who had an
HIV-1 RNA measurement at week 24, the week 8 PBMC
culture result was significantly associated with the probability
of suppression of HIV-1 RNA levels to ?500 copies/ml at
week 24 (P ? 0.001) (Table 7). However, there was no appar-
ent predictive value of the culture result among subjects with a
concentration of HIV-1 RNA at week 8 of ?50 copies/ml
(Table 7). Among subjects with a concentration of HIV RNA
of ?50 copies/ml at week 8, a higher proportion of those with
a negative week 8 culture had subsequent viral load suppres-
sion at week 24 than of those with a positive week 8 culture
(Table 7). However, when adjustment was made for the actual
HIV-1 RNA level at week 8, the culture result was no longer a
significant predictor of subsequent virologic outcome in this
subgroup of patients (P ? 0.31).
We studied the frequency of positive PBMC cultures in a
subset of subjects enrolled in a phase III clinical trial which
compared triple therapy with indinavir and two nucleosides to
dual-nucleoside therapy alone. In this study, we limited our
analyses to subjects who had positive PBMC cultures at base-
line. Cultures were performed in real time with freshly isolated
patient PBMCs according to a standard procedure in which
cultures were defined as positive by using uniform criteria (10).
Of note is that the culture method used did not include CD8
cell depletion. Cultures were held for a minimum of 18 days
before they were determined to be negative, although there
was some variability in the length of time beyond 18 days that
negative cultures were incubated. This study was prompted in
part by reports of negative PBMC culture results, determined
by using standard PBMC culture methods, for subjects with
HIV-1 RNA suppression. Those studies demonstrated that
depletion of CD8 cells from patient and donor PBMCs dra-
matically increased the positive culture rate (13, 17).
Surprisingly, we found that approximately 40% of samples
with a plasma HIV-1 RNA concentration of ?50 copies/ml
yielded a positive PBMC culture. Since cultures with negative
results were not incubated for 28 days at all sites, we believe
that our isolation rate of 40% in these samples is an underes-
timate. We suspect that the higher rate of HIV isolation in our
study than in previously published work is related both to
differences in the patient populations studied and to technical
differences in the standard culture method used. Samples from
patients in our study were obtained for HIV-1 culture between
8 and 40 wks after initiation of treatment, whereas patients
who were previously studied usually had achieved virologic
suppression for longer periods of time. Technical differences in
the culture methods that could have led to higher isolation
rates in our study included the number of patient PBMCs used,
the duration of culture incubation, and our performance of
cultures in real time with freshly isolated patient PBMCs. Pre-
viously published studies have demonstrated that freshly iso-
lated PBMCs give higher culture yields than methods using
TABLE 6. Distribution of positive culture results by the first day on
which a culture was determined to be positive according to
plasma HIV-1 RNA concentration (indinavir arm only)a
Days on which culture
No. of positive cultures (cumulative %) at
plasma HIV-1 RNA concn ofb:
aResults of the standard AMPLICOR MONITOR assay.
bThere were significant differences in the days on which cultures turned
positive between samples with different plasma HIV-1 concentrations (P ?
0.001, GEE test).
cExcludes five samples for which no plasma HIV-1 RNA result was available
for the same time at which the culture was obtained.
TABLE 7. Relationship of suppression of HIV-1 RNA levels
to ?500 copies/ml in subjects at week 24 in indinavir arm with
week 8 HIV-1 RNA concentration and PBMC culture result
% of subjects (no. achieving suppression/total no.
tested) with indicated HIV-1 RNA concn
aP ? 0.31, for testing week 8 culture result (negative versus positive) among
subjects with week 8 RNA level of ?50 copies/ml, after adjusting for week 8 log10
HIV-1 RNA concentration.
2092 DEMETER ET AL.J. CLIN. MICROBIOL.
cryopreserved PBMCs (1, 4, 10). We do not have data on
whether adding a CD8 cell depletion step would further in-
crease the sensitivity of the ACTG culture assay used in our
study. Since few cultures were incubated for more than 28 days,
we also do not know whether the rate of HIV isolation would
be significantly increased by a further increase in the duration
of culture incubation.
We studied in detail the association of treatment and viral
load with culture yield. We found that there were significantly
lower rates of positive cultures in the triple-therapy arm than
in the dual-nucleoside arm. Because the two treatment arms
differed in the frequency of suppression of plasma HIV-1 RNA
levels, we reasoned that samples with lower plasma HIV-1
RNA levels would have a lower rate of positive cultures. We
found that, when our analysis was limited to subjects in the
indinavir triple-therapy arm, the frequency of positive PBMC
cultures was strongly correlated with plasma HIV-1 RNA con-
centration but that a substantial proportion of subjects with
suppressed HIV-1 RNA in plasma still had positive PBMC
The finding that PBMC cultures are positive in approxi-
mately 40% of samples that have plasma HIV-1 RNA levels
below the current limits of detection further supports growing
evidence that reservoirs of replication-competent virus con-
tinue to exist despite the apparent suppression of HIV-1 rep-
lication (2, 5, 7, 17, 18). In addition, at a more pragmatic level,
our findings suggest that obtaining an HIV isolate may be
possible in a substantial proportion of patients with viral load
suppression, without resorting to CD8 cell depletion. We have
demonstrated that positive PBMC cultures from samples with
lower plasma HIV-1 RNA concentrations turn positive at a
later time than those from samples with higher plasma HIV-1
RNA concentrations. Based on the results from this study, the
ACTG consensus protocol for HIV PBMC culture now rec-
ommends a minimum incubation time of 28 days, rather than
the originally recommended 21 days, before a culture is de-
clared negative (6).
Because not all patients in the indinavir arm of the study
developed negative PBMC cultures or achieved virologic sup-
pression, we postulated that we might be able to detect an
association between the PBMC culture result and the subse-
quent virologic outcome. We did find that a negative PBMC
culture result at week 8 was associated with an increased like-
lihood of virologic suppression at week 24. However, the
PBMC culture result did not provide additional predictive
value regarding those subjects with a week 8 HIV-1 RNA
concentration of ?50 copies/ml. This finding suggests that
PBMC culture does not provide predictive information in ad-
dition to that provided by the viral load, although the small
sample sizes limited our power to evaluate this question.
In summary, we have demonstrated that HIV-1 can be iso-
lated from a substantial proportion of patients with viral loads
below the limits of detection of currently available assays, even
when a standard PBMC culture technique is used. Lower viral
loads result in delays in a detectable rise of p24 antigen, and it
is likely that prolonging the incubation time for negative cul-
tures to 28 days will improve the sensitivity of PBMC cultures
to some extent. A negative PBMC culture at 8 weeks of ther-
apy appears to provide some predictive power for subsequent
virologic suppression, although this finding did not persist
when controlling for the week 8 plasma HIV-1 RNA concen-
This work was supported in part by the ACTG and by grants
(AI-27658, AI-38855, AI-38858 [subcontracts 96VC001, 96VC006,
96VC009, 96VC010, 96VC012, 200VC001, 200VC007], AI-27664, AI-
30731, AI-40876, and RR-00044) from the National Institutes of
Health. Supplemental support for some virology studies was provided
by Merck & Company and Roche Molecular Systems. Study medica-
tions were provided by Merck & Company and Glaxo Wellcome Com-
We gratefully acknowledge the subjects and investigators at the
eight AIDS Clinical Trials Units, whose participation made this study
possible. We thank Luis Berrios, Michael Chiulli, and Robin Shepard
for performance of HIV-1 RNA assays and Angela Dexter, Melissa
Kerkau, Derlene Manfredi, Theresa Mescan, Hans Nickstadt, Jana
Parsons, Ruth A. Rhodes, Joan Dragavon, Reggie Sampoleo, Corey
Scherrer, and Kenneth P. Zammit for performance of HIV cultures.
1. Balachandran, R., P. Thampatty, C. Rinaldo, and P. Gupta. 1988. Use of
cryopreserved normal peripheral blood lymphocytes for isolation of human
immunodeficiency virus from seropositive men. J. Clin. Microbiol. 26:595–
2. Chun, T. W., L. Stuyver, S. B. Mizell, L. A. Ehler, J. A. Mican, M. Baseler,
A. L. Lloyd, M. A. Nowak, and A. S. Fauci. 1997. Presence of an inducible
HIV-1 latent reservoir during highly active antiretroviral therapy. Proc. Natl.
Acad. Sci. USA 94:13193–13197.
3. Demeter, L. M., M. D. Hughes, R. W. Coombs, J. B. Jackson, J. Grimes, R. J.
Bosch, S. A. Fiscus, S. A. Spector, K. E. Squires, M. A. Fischl, and S. M.
Hammer. 2001. Predictors of virologic and clinical outcomes in HIV-1 in-
fected patients receiving concurrent treatment with indinavir, zidovudine
and lamivudine (AIDS clinical trials group [ACTG] protocol 320). Ann.
Intern. Med. 135:954–964.
4. Farzadegan, H., D. Imagawa, P. Gupta, M. H. Lee, L. Jacobson, A. Saah, K.
Grovit, C. R. Rinaldo, Jr., and B. F. Polk. 1990. The effect of fresh lympho-
cytes on increased sensitivity of HIV-1 isolation: a multicenter study. J.
Acquir. Immune Defic. Syndr. 3:981–986.
5. Finzi, D., M. Hermankova, T. Pierson, L. M. Carruth, C. Buck, R. E.
Chaisson, T. C. Quinn, K. Chadwick, J. Margolick, R. Brookmeyer, J. Gal-
lant, M. Markowitz, D. D. Ho, D. D. Richman, and R. F. Siliciano. 1997.
Identification of a reservoir for HIV-1 in patients on highly active antiret-
roviral therapy. Science 278:1295–1300.
6. Fiscus, S. A., S. L. Welles, S. A. Spector, and J. L. Lathey. 1995. Length of
incubation time for human immunodeficiency virus cultures. J. Clin. Micro-
7. Furtado, M. R., D. S. Callaway, J. P. Phair, K. J. Kunstman, J. L. Stanton,
C. A. Macken, A. S. Perelson, and S. M. Wolinsky. 1999. Persistence of
HIV-1 transcription in peripheral-blood mononuclear cells in patients re-
ceiving potent antiretroviral therapy. N. Engl. J. Med. 340:1614–1622.
8. Gulick, R. M., J. W. Mellors, D. Havlir, J. J. Eron, C. Gonzalez, D. McMa-
hon, D. D. Richman, F. T. Valentine, L. Jonas, A. Meibohm, E. A. Emini, and
J. A. Chodakewitz. 1997. Treatment with indinavir, zidovudine, and lamivu-
dine in adults with human immunodeficiency virus infection and prior anti-
retroviral therapy. N. Engl. J. Med. 337:734–739.
9. Hammer, S. M., K. E. Squires, M. D. Hughes, J. M. Grimes, L. M. Demeter,
J. S. Currier, J. J. Eron, Jr., J. E. Feinberg, H. H. Balfour, Jr., L. R. Deyton,
J. A. Chodakewitz, and M. A. Fischl. 1997. A controlled trial of two nucle-
oside analogues plus indinavir in persons with human immunodeficiency
virus infection and CD4 cell counts of 200 per cubic millimeter or less. AIDS
Clinical Trials Group 320 Study Team. N. Engl. J. Med. 337:725–733.
10. Hollinger, F. B., J. W. Bremer, L. E. Myers, J. W. Gold, L. McQuay, and the
NIH/NIAID/DAIDS/ACTG Virology Laboratories. 1992. Standardization of
sensitive human immunodeficiency virus coculture procedures and establish-
ment of a multicenter quality assurance program for the AIDS Clinical Trials
Group. J. Clin. Microbiol. 30:1787–1794.
11. Japour, A. J., S. A. Fiscus, J.-M. Arduino, D. L. Mayers, P. S. Reichelderfer,
and D. R. Kuritzkes. 1994. Standardized microtiter assay for determination
of syncytium-inducing phenotypes of clinical human immunodeficiency virus
type 1 isolates. J. Clin. Microbiol. 32:2291–2294.
12. Liang, K. Y., and S. L. Zeger. 1986. Longitudinal data analysis using gener-
alized linear models. Biometrika 73:13–22.
13. Markowitz, M., M. Vesanen, K. Tenner-Racz, Y. Cao, J. M. Binley, A. Talal,
A. Hurley, X. Ji, M. R. Chaudhry, M. Yaman, S. Frankel, M. Heath-Chiozzi,
J. M. Leonard, J. P. Moore, P. Racz, D. F. Nixon, and D. D. Ho. 1999. The
effect of commencing combination antiretroviral therapy soon after human
VOL. 40, 2002HIV ISOLATION IN PATIENTS WITH VIRAL LOAD SUPPRESSION 2093
immunodeficiency virus type 1 infection on viral replication and antiviral Download full-text
immune responses. J. Infect. Dis. 179:527–537.
14. Perelson, A. S., P. Essunger, Y. Cao, M. Vesanen, A. Hurley, K. Saksela, M.
Markowitz, and D. D. Ho. 1997. Decay characteristics of HIV-1-infected
compartments during combination therapy. Nature 387:188–191.
15. Staszewski, S., J. Morales-Ramirez, K. T. Tashima, A. Rachlis, D. Skiest, J.
Stanford, R. Stryker, P. Johnson, D. F. Labriola, D. Farina, D. J. Manion,
N. M. Ruiz, and the Study 006 Team. 1999. Efavirenz plus zidovudine and
lamivudine, efavirenz plus indinavir, and indinavir plus zidovudine and lami-
vudine in the treatment of HIV-1 infection in adults. N. Engl. J. Med. 341:
16. Wong, J. K., H. F. Gunthard, D. V. Havlir, Z. Q. Zhang, A. T. Haase, C. C.
Ignacio, S. Kwok, E. Emini, and D. D. Richman. 1997. Reduction of HIV-1
in blood and lymph nodes following potent antiretroviral therapy and the
virologic correlates of treatment failure. Proc. Natl. Acad. Sci. USA 94:
17. Wong, J. K., M. Hezareh, H. F. Gunthard, D. V. Havlir, C. C. Ignacio,
C. A. Spina, and D. D. Richman. 1997. Recovery of replication-competent
HIV despite prolonged suppression of plasma viremia. Science 278:1291–
18. Zhang, L., B. Ramratnam, K. Tenner-Racz, Y. He, M. Vesanen, S. Lewin, A.
Talal, P. Racz, A. S. Perelson, B. T. Korber, M. Markowitz, and D. D. Ho.
1999. Quantifying residual HIV-1 replication in patients receiving combina-
tion antiretroviral therapy. N. Engl. J. Med. 340:1605–1613.
2094 DEMETER ET AL.J. CLIN. MICROBIOL.